This Competitive Revision application is in response to NOT-OD-09-058 "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications." We request competitive supplement to our NIH grant R21AG032567 entitled "Role of p62 in Protein Aggregation and Neurodegeneration in ALS." Protein aggregates containing mutant copper-zinc superoxide dismutase (SOD1) are a hallmark of familial amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), an age-related neurodegenerative disease. P62/Sequestosome 1 (referred as p62 in this proposal) is a multifunctional protein involved in both of the two major protein degradation mechanisms: ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway. The central hypothesis to be tested in the parent R21 project is that p62 can recognize misfolded mutant SOD1 and that p62 can ameliorate the mutant SOD1 induced toxicity by shuttling such misfolded proteins to UPS and/or autophagy. We previously reported that p62 was co-localized with mutant SOD1 in the protein aggregates in spinal motor neurons in G93A SOD1 transgenic mice. Co-immunoprecipitation experiments showed that p62 specifically recognized mutant SOD1, but not the WT protein. Three specific aims were designed to test the hypothesis at the time. Aim 1 was to map the domains of p62 essential for recognizing and interacting with mutant SOD1. Aim 2 was to determine whether and how p62 mediated the autophagy activation induced by mutant SOD1. Aim 3 was to study how p62 would influence protein aggregation and ALS disease progression in vivo using p62 KO mice. We have largely accomplished the Aims 1 and 2 and are currently performing experiments proposed in Aim 3. This Competitive Revision is built on the progress made in the R21 project and the new preliminary data suggesting that HDAC6 is likely an adaptor protein between p62 and mutant SOD1. In addition, HDAC6 has been shown to play a critical role in autophagy activation and stress response. We thus propose to expand the parent R21 project to test a new hypothesis that HDAC6 is an adaptor protein mediating mutant SOD1-p62 interaction, regulate the autophagic degradation of mutant SOD1, and mediate the stress response induced by mutant SOD1. Three new specific aims are proposed to test this hypothesis. Aim 1 is to dissect the detailed mechanism how HDAC6 functions as an adaptor between mutant SOD1 and p62. Aim 2 is to determine the role of HDAC6 in aggregation and autophagic degradation of mutant SOD1. Aim 3 is to study the significance of HDAC6 and p62 in mutant SOD1 induced stress response. Results from this study will provide invaluable insights into the role of HDAC6 and p62 in protein aggregation and neurodegeneration in ALS, which will result in better understanding of ALS etiology and potential discovery of new therapeutic target for ALS treatment. Relevance to the American Recovery &Reinvestment Act (ARRA): This application will create two new scientific researcher positions as soon as it is funded. Purchase of necessary equipment, supplies and reagents for the proposed research will also stimulate the economy as intended by the ARRA. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is an age-related neurodegenerative disorder characteristic of the preferential motor neurons death. Mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial ALS cases. Protein aggregates containing mutant SOD1 are a hallmark of familial ALS. P62/Sequestome 1 (referred as p62 in this proposal) is a multifunctional protein involved in both of the two major protein degradation mechanisms: ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway. The parent R21 was to test the hypothesis that p62 could recognize misfolded mutant SOD1 and ameliorate the mutant SOD1 induced toxicity by shuttling such misfolded proteins to the UPS and/or autophagy. We have largely accomplished the Aims 1 and 2 proposed in the parent R21 and are currently performing experiments proposed in Aim 3. Based on the progress and new preliminary results, we propose to test the significance of an adaptor protein named histone deacetylase 6 (HDAC6) in mutant SOD1 protein degradation, aggregation and neurotoxicity. The specific hypothesis to be tested in the expanded project is that HDAC6 is an adaptor protein mediating mutant SOD1-p62 interaction, regulates the autophagic degradation of mutant SOD1, and mediates the stress response induced by mutant SOD1. Results from this study will provide new insights into the role of HDAC6 and p62 in protein aggregation and neurodegeneration in ALS, which will result in better understanding of ALS etiology and potential discovery of new therapeutic target for ALS treatment. Moreover, this application will create two new scientific researcher positions as soon as it is funded. Purchase of necessary equipment, supplies and reagents for the proposed research will also stimulate the economy as intended by the American Recovery &Reinvestment Act.